Therapeutic hyperthermia is the production of super-physiological temperatures for treatment of disease. Hyperthermia is currently being investigated as an adjuvant for many cancer therapies, (e.g. prostate cancer) including radiation and photodynamic therapy. The oxidative stress theory of heat shock has the following components: heat shock can produce free radicals and related oxidants; these species can cause part of the cellular injury produced by heat; and cellular injury can be tolerated if protective proteins are induced. The research proposed in this application is designed to test the oxidative stress theory of heat shock. We hypothesize that cells subjected to hyperthermia (e.g., 41-45 C) will produce an increased flux of free radicals. Thus, using cultured cells we will: detect and identify these free radicals using electron paramagnetic resonance; examine the overall flux or oxidants in cells; and examine the response of cells to these oxidants. We hypothesize that elevated levels of the antioxidant enzymes superoxide dismutase (SOD) and/or glutathione peroxidase (GPx) will lead to thermotolerance, whereas depressed levels of SOD and/or GPx will lead to heat sensitivity. We will test this hypothesis by using molecular biology techniques to alter the antioxidant enzyme level in cells and then test their thermotolerance. For those cells capable of generating nitric oxide, we hypothesize that heat will result in increased production of nitric oxide, which under certain circumstances, can be cytotoxic. We will examine the ability of cultured endothelial (and other appropriate) cells to generate nitric oxide in response to hyperthermia and determine if this nitric oxide is detrimental. If ROS are important, then we would predict that catalytic iron would increase in heat-stressed cells, thereby amplifying the oxidations initiated by heat treatment. We will examine the role of this iron in producing cell toxicity during hyperthermia and determine if modulation of catalytic iron levels will alter the thermotolerance of cells. This proposed research program is designed to test the hypothesis that production of free radicals and related oxidants is an important aspect of the detrimental effects of hyperthermia.